Hepatitis C virus (HCV) is the major etiological agent of post-transfusion non-A, non-B hepatitis. Genetic heterogeneity of HCV has been shown to have significant clinical and diagnostic implications. Therefore, it is of importance to develop reliable, rapid and cost-effective assays for HCV genotyping. A novel method that consisits of RT-PCR, followed by single strand conformation polymorphism analysis (SSCP) of the variable region of the HCV genome, was used for the determination of HCV genotypes. PCR products are denatured, and then they are applied to a nondenaturing polyacrylamide gel (PAGE), followed by ethidium bromide staining. Serum samples with HCV genotypes I, II, III, and IV (provided by Dr.Purcell, NIH), determined by sequencing, were used to develop specific SSCP patterns for each of the genotypes. Sera from 4 HCV RNA positive patients, and serum from an experimentally HCV infected chimpanzee were tested. Three out of the four patients and the chimpanzee were identified with genotype II, and one patient showed a mixed infection of genotypes I, II, and III. These results, which were confirmed by a genotype-specific primer method, demonstrate that RT-PCR-SSCP is a reliable, fast and easy to perform method for the determination of HCV genotypes.